1. Field of the Invention
The present invention relates to an apparatus and a method for determining the functioning properties of cells by measuring a fine response by the cells contained in a cell suspension when a shear stress is applied to the cell suspension. More particularly, the present invention relates to an apparatus and a method in which, for example, a shear stress is applied to a platelet suspension, and the fine aggregation of the platelets, the variation of the forms of the platelets and the release reaction of the platelets, etc. due to the applied shear stress are continuously measured with a high sensitivity. The present invention can be broadly used for diagnosis and treatment of congenital diseases such as thrombasthenia and von Willebrand disease and treatment of acquired diseases such as myocardial infarction, thrombosis and arteriosclerosis, which cause platelets to function abnormally. Further, an apparatus according to the present invention can be also utilized for development of medicines which are intended to treat platelet abnormalities.
2. Description of the Prior Art
Various apparatuses which determine the functioning properties of cells using a cell suspension have been developed. Particularly, the apparatus designed by Born and O'Brien (Born, G. V. R.: Nature, page 194, 927 (1962), and O'Brien, J. R.: J. Clin. Path. page 15, 446 (1962)) and the apparatus designed by Salzman (Salzman, E. W.: J. Lab. Clin. Med., page 62, 724(1963)) are known as apparatuses which can determine functioning properties of platelets, and these apparatuses are broadly used for clinical examinations.
The former apparatus (hereinafter called the first conventional apparatus) is used for examining the aggregation properties of platelets. This apparatus comprises an unit which includes a cylindrical glass container having the diameter of about 5 mm for measuring the transparency of a platelet suspension and a recorder which converts the measured transmittance to a corresponding electric signal and records the aggregation curve with a pen. In this apparatus, the platelet suspension of 0.2-0.3 ml which is separated from blood by a centrifugal separator is contained in a cylindrical glass container, and an aggregating agent such as adenosine diphosphate, collagen, epinephrine, ristocetin or thrombin is added to it. When the platelet suspension is stirred by a magnetic stirrer previously placed in the container, the platelets are rapidly formed into flocks and the measured transmittance of the platelet suspension increases. The variation of this transmittance can be continuously recorded as the aggregation curve of the platelets.
The latter apparatus (hereinafter called the second conventional apparatus) is used for examining the adhesive properties of platelets. This apparatus utilizes the characteristic of platelets for adhering to a glass surface. In this apparatus, blood is passed through a tube filled with glass beads, and the variation of the number of platelets before the passage as compared to after the passage is measured. The apparatus comprises a tube having an inner diameter of about 2 mm and a length of about 15 cm and filled with glass beads having a diameter of 0.3-0.4 mm and an empty tube for taking a sample of blood. The respective ends of the two tubes are connected to each other via a three-way cock having a needle for taking blood. Plastic syringes are connected to the respective unconnected ends of both the tubes. Further, an aspirator is connected to the two syringes, and blood is taken thereinto at a constant speed.
An academic publication reports the variation in properties of platelets caused when a shear stress of about 1 dyne/cm.sup.2 is applied to a platelet suspension and is continuously recorded (Klose, H. J., Rieger, H. and Schid-Schonbein, H.: Thrombosis Res., page 7, 261 (1975)). An experimental apparatus (hereinafter called the third conventional apparatus) is disclosed in this report. The apparatus comprises a transmissible concave bath and a rotor having a conical surface facing the inner bottom surface of the bath at an angle of 3.degree. and having a diameter of 5 cm. Two optical fibers are provided on the outer side surface of the bath for projecting a ray into the platelet suspension between the rotor and the inner bottom surface of the bath in the direction of the flow of the platelet suspension via one of the optical fibers and for detecting the transmitted ray from the inside of the bath via the second of the optical fibers. A halogen-lamp-stabilized light source is connected to the end of the optical fiber used for projecting the ray, and a photo detector connected to a recorder is connected to the end of the optical fiber used for detecting the transmitted ray.
Moreover, an apparatus which is improved over the third conventional apparatus (hereinafter called the fourth conventional apparatus) is reported (Rieger, H., Baier, H., Schroder, H., Wurzinger, L., Schid-Schonbein, H. and Blasberg, P.: Thrombosis Res. page 17, 589 (1980)). This fourth conventional apparatus has a structure, similar to a double cylinder type rotational viscometer, which comprises an inner cylinder having a light source and an outer cylinder rotatably provided around the inner cylinder. Although a double cylinder type rotational viscometer, generally, has a defect in that a secondary flow due to Taylor vortex is liable to occur, this defect is overcome in the fourth conventional apparatus by rotating the outer cylinder.
Furthermore, an apparatus similar to the third conventional apparatus (hereinafter called the fifth conventional apparatus) is reported (Frojmovic, M. M.: Biorheology, page 12, 193 (1975)). In this apparatus, an optical path length in a platelet suspension is reduced up to about 1 mm by disposing a light source and a photo detector in a direction perpendicular to the direction of the flow of the platelet suspension.
In the first conventional apparatus wherein functions of platelets are determined from the aggregation curve obtained by adding aggregating agents to the platelet suspension, there is a problem in that the stimulus applied to the platelets is not physiological. For example, in a clinical examination using this apparatus, the concentration of adenosine diphosphate in the platelet suspension is usually controlled from 2 .mu.M to 10 .mu.M, but it is unknown at all whether the adenosine diphosphate of such a high concentration causes the aggregation of platelets in an organism or not. Moreover, ristocetin which is one of the aggregation agents does not exist in an organism. Further, in spite of the fact that it is reported by many researchers that platelets are activated by the influence due to the flow of blood, the factor presenting the flow state of blood is defined only by the rotational speed of the stirrer which is generally not consistent and is not accurately determiable. Furthermore, since the optical path length in the platelet suspension is only about 5 mm, the variation of the amount of the transmitted ray due to a fine aggregation of the platelets in response to a fine stimulus in an organism cannot be detected. Thus, since the first conventional apparatus has the above problems, the results obtained by the apparatus do not accurately identify clinical symptoms at many times in spite of the broad use of the apparatus in clinical examinations.
In the second conventional apparatus wherein the adhesive characteristics of platelets for adhering to a glass surface is utilized, since the nonphysiological phenomenon of adhesion to a glass surface occurs, there are problems similar to those in the first conventional apparatus. Moreover, since the sample flows between glass beads in this apparatus, the flow state, which greatly influences the adhesive characteristics of platelets, is quite different from the flow state in an organism. Therefore, the results obtained by the apparatus frequently do not accurately represent the true properties of platelets.
On the other hand, the common characteristic of the third, fourth and fifth conventional apparatuses is that the flow state of a platelet suspention can be specified by shear stress which is a generic factor. Since the reactions of platelets play an important part in diseases such as myocardial infarction, thrombosis and arteriosclerosis, much research and many discussions have been conducted for many years as reported in many scientific and academic reports and documents, using these apparatuses and other apparatuses which can specify the flow state of a platelet suspension. Despite this fact, the third, fourth and fifth conventional apparatuses have not yet been capable for clinical examinations in comparison with the first and second conventional apparatuses. The reason is that, since the third, fourth and fifth conventional apparatuses have the following problems, the mechanism of the aggregation and reaction properties of platelets due to the shear stress has not been elucidated and the correlation between the aggregation and reaction of platelets and diseases has not been clarified.
First, the third conventional apparatus aims to measure the aggregation of platelets under the condition of a low shear stress such as about 1 dyne/cm.sup.2 and uses a rotor having a conical surface which faces the inner bottom surface of the bath at an angle of 3.degree.. Therefore, the uniform shear stress, which can be applied to the platelet suspension having a viscosity of about 1 centipoise by use of this apparatus, is about 5 dyne/cm.sup.2 at the highest. When a shear stress higher than the above value is applied to the platelet suspension, the flow state of the suspension becomes turbulent, and the applied stress becomes nonuniform. Moreover, in this apparatus, since the light having a broad wave length distribution emitted from a halogen lamp light source is projected into the platelet suspension without use of an optical filter or a spectroscope, the transmittance of the rays that are detected is liable to be influenced by the absorption of the rays due to the protein in the blood and/or the scattering of the rays due to the lipid in the blood. Furthermore, the relationship between the size of the particles and the scattering of light varies in a complicated manner according to the wave length of the light in a big particle dispersion system such as a platelet suspension. Therefore, when the light having such a wave length distribution is directly projected into the platelet suspension, a fine aggregation of the platelets due to the shear stress cannot be detected. The fourth conventional apparatus improves the defect of the third conventional apparatus by reducing the optical path length by using a structure of double cylinders. In this apparatus, however, since the optical path length is almost the same as or smaller than that in the first conventional apparatus, a large aggregation of the platelets can be measured, but a fine aggregation of the platelets due to a very small physiological stimulus cannot be measured.
The optical path length in the fifth conventional apparatus is very small, i.e. about 1 mm. Therefore, this apparatus is quite unsuitable for measuring a fine variation of platelets.